Human Luteinized Granulosa Cells Research Articles

Apoptosis of cultured granulosa-lutein cells is reduced by insulin-like growth factor I and may correlate with embryo fragmentation and pregnancy rate

To correlate apoptosis of cultured human granulosa-lutein cells (GL cells) with the outcome of IVF (embryo fragmentation and pregnancy rate) and to study the effect of insulin and insulin-like growth factor I (IGF-I) on apoptosis. In vitro assays. University laboratory and private IVF center. Eighty-one women undergoing IVF. Purified human GL cells from pooled follicles were cultured for 48 hours in serum-free media with or without insulin and IGF-I. Cumulus cells and mural GL cells were evaluated separately. Detection of apoptosis by using caspACE FITC-VAD-FMK, a fluorescent in situ marker for activated caspases; embryo fragmentation; and pregnancy. Age younger than 38 years and successful pregnancy were associated with less apoptosis (33.0% +/- 17.2% vs. 43.2% +/- 18.0% and 30.2% +/- 14.0% vs. 40.4% +/- 19.5%, respectively). There was a linear correlation between embryo fragmentation and GL cell apoptosis. Insulin-like growth factor I decreased apoptosis in a dose-dependent fashion. A statistically significant effect (17% decrease) was reached at a dose of 10 nM. Insulin (10 nM) caused a small (8%) decrease in apoptosis, but this effect did not reach statistical significance. Cumulus cells consistently had <3% apoptosis. [1] Apoptosis of cultured GL cells may be associated with IVF outcome and ovarian reserve and [2] IGF-I decreases apoptosis of cultured GL cells.

Carbaryl inhibits basal and FSH-induced progesterone biosynthesis of primary human granulosa-lutein cells

Carbaryl is known to impede female reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into the mechanisms, this study was conducted to raise fresh concerns about the potential effects of carbaryl on steroidogenesis by primary human granulosa-lutein cells (hGLCs) and explore the possible nature of this action. hGLCs were co-incubated with various concentrations of carbaryl at 0, 1, 5, 25, 125 μmol/L for 24 h to examine effects of this carbamate pesticide on progesterone accumulation. We observed that the carbaryl inhibited basal and FSH-induced progesterone production in a dose-dependent manner. We also investigated the effects of carbaryl on 22( R)-hydroxycholesterol (22 R-HC)-stimulated progesterone yield, basal and FSH-stimulated StAR gene expression and cyclic adenosine monophosphate (cAMP) production, as well as forskolin (non-specific activator of adenylyl cyclase)-induced progesterone and cAMP production of hGLCs. We found that the decreased progesterone biosynthesis was accompanied with a reduced cAMP abundance on both basal and FSH-induced condition. Furthermore, our results demonstrated that the 22 R-HC could remove the carbaryl-induced restraint of progesterone biosynthesis, suggesting that carbaryl caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that carbaryl inhibited the gene expression of steroidogenic acute regulatory protein (StAR). In addition, the inhibitory effects of carbaryl on progesterone and cAMP production were completely reversed by addition of forskolin to the cell culture, which indicated a repaired site on the upstream components of adenylate cyclase or adenylate cyclase per se by carbaryl in the cAMP-mediated signal pathway. All the effects mentioned above were not due to a detrimental action of carbaryl on cell viability by MTS assay. In conclusion, carbaryl may inhibit steroidogenesis, at least in part, by obstructing the delivery of cholesterol over mitochondrial membranes and attenuating cAMP generation.

Environmental toxicants and effects on female reproductive function.

One of the most toxic substances known to humans, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), is also highly pervasive in the environment. It is created naturally in volcanic eruptions and forest fires, and anthropogenically in waste incineration, chlorination processes and certain plastics manufacture. From reports of large industrial and other accidents, or from experimental studies, dioxin exposure has been correlated in animal models and/or humans with chloracne of the skin, organ cancers, hepatotoxicity, gonadal and immune changes, pulmonary and other diseases such as diabetes, skewing of the sex ratio, and infertility. We have demonstrated that the aromatic hydrocarbon receptor (AHR) that binds dioxin in tissues is localized in zebrafish, rat and rhesus monkey (Macaca mulatta) ovaries and in rat and human luteinizing granulosa cells (GC) (among other tissues), that labeled dioxin is specifically localized to granulosa cells of the ovarian follicle as observed by autoradiography, and that incubations of GC or ovarian fragments with environmentally relevant concentrations (fM to nM) of dioxin inhibit estradiol secretion significantly. Our experiments show that in human, non-human primate, rat, trout, and zebrafish ovarian tissues, dioxin inhibits estrogen synthesis at some level of the steroid biosynthetic pathway, most likely by inhibiting transcription of mRNAs for or activity of side-chain cleavage (Cyp11a1 gene) and/or aromatase (Cyp19a1 gene) enzymes, or conceivably other steroidogenic enzymes/factors. Such an untoward effect on estrogen synthesis in females exposed to dioxin environmentally may predispose them to defects in aspects of their fertility.

TCDD Increases Inhibin A Production by Human Luteinized Granulosa Cells In Vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic of the halogenated dioxins and one of the most poisonous substances known to man. The major toxic effects of TCDD on reproduction are decreased fertility and diminished ability to maintain a pregnancy. Granulosa cells obtained from hormonally stimulated women participating in an in-vitro fertilization program were cultured with 3.1 femtomolar, 3.1 picomolar and 3.1 nanomolar TCDD. While inhibin B production was not altered, inhibin A production increased significantly after 4 hours of exposure to both nanomolar and micromolar TCDD concentrations. By 8 hours of exposure to these concentrations of dioxin, human luteinizing granulosa cells exhibited a pronounced increase in inhibin A, nearly quadrupling secretion from unexposed control cells. TCDD continued to increase inhibin A secretion at the picomolar concentration at 24 and 36 hours. It is conceivable that TCDD may act at the ovary to augment inhibin A secretion, thereby reducing FSH-stimulable estrogen secretion by granulosa cells.

ABR-014 Study of the responsive genes of human luteinized granulosa cells to growth hormone (GH)

ABR-014 Study of the responsive genes of human luteinized granulosa cells to growth hormone (GH)

Rapid action of pesticides on cytosolic calcium concentrations in cultures of human umbilical vein endothelial cells

Persistent metabolites of pesticides such as p, p′-DDE, at environmentally relevant concentrations, have been shown to have a rapid effect on intracellular calcium [Ca 2+] i concentrations in human granulosa-lutein cells. Since endocrine disrupting substances can be transferred from the maternal circulation to the fetus the present study examined whether the pesticides, kepone, o, p-DDE, p, p′-DDE and methoxychlor, could alter cytoplasmic calcium [Ca 2+] cyt concentrations in human umbilical vein endothelial (HUVE) cells. Cultured HUVE cells were loaded with Fura-2 AM and changes in [Ca 2+] cyt of single cells were studied using a dynamic digital Ca 2+ imaging system. Kepone and methoxychlor consistently increased [Ca 2+] cyt concentrations, similar to the effects of estradiol and progesterone. p, p′-DDE increased [Ca 2+] cyt concentrations in 80% of experiments whereas o, p-DDE stimulated its increases in 42%. Estrone, estriol, pregnenolone and cortisol were not effective. These results demonstrate that pesticides can have a rapid effect on HUVE cells probably through a nongenomic mechanism of action.

Calcium-dependent up-regulation of mitochondrial electron transfer chain gene expressions in human luteinized granulosa cells

To evaluate the transcription and translation ability of mitochondria in terminally differentiated granulosa cells, these cells were incubated with ionic calcium. Prospective laboratory research. In vitro fertilization laboratory in a university hospital. Granulosa cells were harvested from 50 female patients undergoing IVF. Analysis of mitochondrial gene expression by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and of mitochondrial-encoded proteins by Western blot. Comparison of the RNA expression levels of genes including cytochrome c oxidase subunit I (COX I), adenosine triphosphate synthase 6 (ATPase 6), flavoprotein, and succinate-ubiquinone oxidoreductase, and protein levels of COX I and flavoprotein in different calcium ion treatment groups. There were dose-dependent increases in RNA expressions of the four genes analyzed from granulosa cells cultured in a serial concentration of calcium ions. This effect was abolished when cells were preincubated with the extracellular calcium-chelating agent, Ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The effect of ionic calcium on both the nuclear- and mitochondrial-encoded subunits also was determined. Expression levels of mitochondrial transcription factor A in RNA significantly increased in granulosa cells that were exposed for 24 and 48 hours to 0.5 and 1 microM A23187. The present study is the first report to present calcium-dependent increases in the transcription and translation levels of both nuclear-encoded and mitochondrial-encoded mitochondrial respiratory enzyme subunits and also indicates that mitochondrial transcription factor A is involved in mitochondrial biogenesis.

Rapid Effects of Pesticides on Intracellular Calcium Changes in Human Granulosa-Lutein Cells

Rapid Effects of Pesticides on Intracellular Calcium Changes in Human Granulosa-Lutein Cells

Effect of Angiotensin II (AngII) on Apoptosis of Human Granulosa-Lutein Cells: A Correlation With IVF Outcome

To study the effect of Ang II on apoptosis of cultured human granulosa-lutein cells (GL cells) and correlate this effect with the outcome of IVF Detection of apoptosis in GL cells using a fluorescent in situ marker after 48 hour culture in the presence or absence (control) of Ang II and its receptor antagonist saralasin 37 IVF cycles were analyzed. Three patients in which the embryo transfer was cancelled were excluded from this analysis. Ovulation induction was performed using recombinant FSH combined with recombinant LH or HMG. The fertilization method was intra-cytoplasmic sperm injection (ICSI) in all cases. GL cells were collected from pooled follicular fluid after oocyte recovery. Cells were washed and further purified using a 50 % Percoll gradient and anti-CD45 coated magnetic beads. After 48 hour culture without serum in the presence or absence of 10-11, 10-9 and 10-7 M Ang II and saralasin 10-5M, the percentage of apoptotic cells was determined under a fluorescent microscope after staining with 10 μM caspACE FITC-VAD-FMK (a fluorescent marker for activated caspases) and 5 μg/ml propidium iodide. Statistical analysis was carried out using the Mann-Whitney U test. All doses of AngII had an effect on apoptosis that was blocked by the angiotensin receptor antagonist saralasin. Ang II 10-9 and 10-11M increased apoptosis in patients who became pregnant and decreased apoptosis in patients who did not conceive (Table 1). AngII 10-7M increased apoptosis in most patients (14/18) and no correlation with the outcome of the cycle was found (p=0,374). Tabled 1 A correlation was found between pregnancy and increased apoptosis after culture of human granulosa lutein cells in the presence of 10-9 and 10-11M AngII. It is now necessary to assess whether the relationship of AngII and apoptosis is causative or the result of intervening actions by AngII.

Differential Regulation of Vascular Endothelial Growth Factor (VEGF-A) Secretion by Insulin and Insulin-Like Growth Factors (IGF) in Human Luteinizing Granulosa Cells (LGC) Following Controlled Ovarian Stimulation (COS) in a Polycystic Ovarian (PCOS) Population

To investigate the role of IGF-I and -II in the physiological regulation of VEGF-A production by LGC from preovulatory follicles in PCOS patients in the presence and absence of LH. A prospective, comparative, experimental study In an IRB approved protocol, LGC were collected via follicular aspiration from women (n=6) with PCOS undergoing COS for in-vitro fertilization. The diagnosis of PCOS was established using the ASRM/ESHRE consensus guidelines. The mean age of the study population was 30.2 ± 0.82 years. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 mu;g/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin, 25 mu;g human LDL. Cells were incubated with 0, 1, 10, and 100 ng/ml of r-h IGF-1, IGF-II, and insulin (Sigma) in the presence or absence (control) of 100 ng/ml LH (Ares Serono). LGC were cultured for 72 hours and media were collected daily and assayed for free VEG-F (Quantikine VEGF ELISA, R&D Systems) and progesterone (P). After 3 days, wells were fixed with a Crystal Violet stain and analyzed for DNA content to estimate cell numbers. Data were normalized to DNA content and analyzed using a linear regression model, a mixed effects model, and adjusted using the Bonferroni method for multiple comparisons Exposure to IGF-I, IGF-II and insulin typically increased (p<0.05) DNA content in cell cultures, necessitating the normalization of P and VEGF levels to DNA content. The presence of 1-100 ng/ml IGFs or insulin did not alter control or LH-stimulated P levels during 3 days of culture. Under control conditions, LGCs secreted detectable levels of VEGF-A throughout culture. Addition of IGF-I, IGF-II or insulin (1-100ng/ml) caused a dose-dependent increase (p<0.05) in VEGF levels by day 2-3 of culture. In the absence of LH, physiological levels (1-10 ng/ml) of IGF-I or -II did not increase VEGF concentrations, whereas the same levels of insulin tended to increase (p=0.02) VEGF at 72 hrs. In the presence of LH, again there was no effect of 1-10 ng/ml IGF-I or II at 2-3 days, but insulin significantly (p<0.001) increased VEGF concentrations during this interval. Notably, these results are markedly different from those we reported earlier using LGC from non-PCOS patients during IVF protocols (Fertil Steril Supp 3, 80: S278, abstract P477, 2003): (1) IGFs and insulin did not markedly alter DNA content during 3-day culture, and (2) at 1-10 ng/ml, both IGF-I and -II, but not insulin, increased VEGF levels in the culture media. Exposure to both insulin and IGFs, as well as LH, for greater than 24 hours promotes VEGF-A secretion by LGC from PCOS women. Thus, both endocrine and local factors play a role in regulating VEGF production by the ovulatory, luteinizing follicle. However, there appear to be differences in responses between cells from non-PCOS and PCOS women, with the latter more sensitive to insulin and less sensitive to IGFs in terms of granulosa cell VEGF-A production, particularly in the presence of LH. Insulin and IGFs also selectively promoted cell proliferation of granulosa cells from PCOS women. Whether these characteristics reflect inherent differences in the maturing follicle, or the responses from treatment during COS in women with PCOS remains to be established.

Effects of the insecticide amitraz, an α2-adrenergic receptor agonist, on human luteinized granulosa cells

Amitraz, an insecticide used to prevent tick and mite infestation of cattle, crops and dogs, is an alpha2-adrenergic receptor agonist that inhibits GnRH release and the ovulatory LH surge in rats. Noradrenalin, the physiological ligand for adrenergic receptors, inhibits progesterone production by IVF-derived granulosa cells, but the effects of amitraz are unknown. Luteinized granulosa cells obtained from women undergoing ovarian stimulation were exposed to amitraz (1, 10, 50, 100 microg/ml) for 2-72 h, and to amitraz (50 microg/ml) +/- hCG or the specific alpha2-adrenergic receptor antagonist yohimbine, for 6 h. Cell numbers were determined by 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay and hormone production by radioimmunoassay. Amitraz 10 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone production to 58 +/- 8% (p < 0.01, 24 h, n = 6) of control values. Amitraz (100 microg/ml) was cytotoxic and caused a corresponding reduction in hormone production. Amitraz 50 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone per cell production to 82 +/- 6% of control values after 6 h. This was prevented by 0.2 mmol/l yohimbine. Exposure to amitraz 50 microg/ml for 6 h exposure abolished hCG-stimulated progesterone production but not estrogen production. Amitraz inhibited basal and hCG-stimulated progesterone but not estrogen production. The inhibitory action of amitraz and its antagonism by yohimbine suggest that alpha2-adrenergic receptors are expressed by luteinized human granulosa cells.

Expression and regulation of Sprouty-2 in the granulosa-lutein cells of the corpus luteum

Growth factor signalling has important modulatory roles in the process of human follicular growth, oocyte maturation and corpus luteum (CL) formation. Recently, Sprouty-2, an inhibitor of receptor tyrosine kinase (RTK) signalling pathway was advocated as a marker of oocyte competence in the bovine ovary. We sought to study Sprouty-2 expression and regulation in the human ovary. RT-PCR was used to detect Sprouty-2 mRNA in human granulosa-lutein cells (GLC) collected from follicular aspiration of IVF patients. The effect of epidermal and fibroblast growth factors (EGF and FGF) on Sprouty-2 mRNA expression in GLC was studied using quantitative real-time PCR. Immunohistochemistry was performed on cultured GLC, human CL and stimulated rat ovary sections. Sprouty-2 mRNA was expressed in human GLC. EGF and basic FGF, but not FGF4 and FGF10, increased Sprouty-2 mRNA expression in GLC. The Sprouty protein was localized to GLC of early and late human CL but not to the theca cell layer. Immunostaining of developing rat CL confirmed the temporal and spatial expression of Sprouty in humans. The detection of Sprouty-2 mRNA and protein in human GLC may suggest a role for Sprouty-2 during the final stages of follicle maturation and CL formation.

The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12-13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11-13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.

Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells

The present study examined whether the sex steroids, estradiol and progesterone, could alter cytoplasmic calcium concentrations ([Ca(2+)](cyt)) in human granulosa-lutein cells. Human granulosa cells were obtained at the time of oocyte retrieval for IVF and cultured for 3-7 days. Cells were loaded with Fura-2 AM and changes in [Ca(2+)](cyt) of single cells were studied using a dynamic digital Ca(2+) imaging system. Both estradiol and progesterone stimulated elevations of [Ca(2+)](cyt) in Ca(2+)-containing medium within seconds of exposure of the granulosa-lutein cells to the steroid, but only estradiol caused an increase in [Ca(2+)](cyt) in Ca(2+)-free medium. Both ICI-182780 and RU 486 stimulated [Ca(2+)](cyt) increases and inhibited the effects of estradiol and progesterone, respectively. Tamoxifen also induced transient increases in [Ca(2+)](cyt) concentrations but inhibited the effects of both estradiol and progesterone. The inhibitory effects of tamoxifen, ICI-182780 and RU 4486 on [Ca(2+)](cyt) responses to estradiol and progesterone could be reversed with higher concentrations of estradiol and progesterone, respectively. The [Ca(2+)](cyt) effects induced with tamoxifen could not be eliminated by prior treatment with RU 486 or ICI-182780. These results provide strong evidence that both estradiol and progesterone as well as the steroid antagonists, tamoxifen, RU 486 and ICI-182780, can act on human granulosa-lutein cells through a non-genomic mechanism.

2,3,7,8-Tetrachlorodibenzo- p-dioxin induces CYP1B1 expression in human luteinized granulosa cells

The environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is a reproductive toxicant in multiple species; however, mechanisms and direct ovarian effects are poorly understood. DNA microarrays were used to characterize gene expression profiles of human luteinized granulosa cells (HLGCs) exposed to TCDD in primary cultures. Exposure to 10 nM TCDD for 24 h induced a significant increase in CYP1B1, while few other genes responded. TaqMan PCR and Western immunoblotting demonstrated that induction was dose-dependent. Additionally, the microsomal form of catechol- O-methyltransferase (COMT) was highly expressed in HLGCs, along with only fractional amounts of the soluble form. This is the first report of CYP1B1 and COMT expression, and CYP1B1 induction, in cells from the human ovary. The role of CYP1B1 in the oxidative metabolism of estrogens and potential generation of DNA adducts in the ovary may have significant consequences for oocyte quality, corpus luteum function, and ovarian carcinogenesis.

Deleted Mitochondrial DNA in Human Luteinized Granulosa Cells

The rearrangement of mitochondrial DNA in luteinized granulosa cells was determined in order to evaluate the fertilization capacity of oocytes and the development of embryos. Multiple deletions of mtDNA were found in luteinized granulosa cells from in vitro fertilization (IVF) patients. The 4977-base pair (bp) deletion was the most frequent deletion found in human granulosa cells. No significant difference was noted between mtDNA deletions of granulosa cells based on the fertilization capacity of oocytes and the development of embryos. To determine the relationship of proportions of mtDNA rearrangements with the aging process, granulosa cells were grouped into three different cohorts according to maternal age: younger than 32 years, between 32 and 37 years, and older than 37 years. No statistical correlation was noted between patient age and the frequency of occurrence of multiple mtDNA deletions. However, an increase in granulosa cell apoptosis was associated with an increase in mtDNA deletions. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We concluded that the accumulation of rearranged mtDNA in granulosa cells might not interfere with fertilization of human oocytes and further embryonic development; it was, however, associated with apoptosis processes.

Expression of steroid receptors, their cofactors, and aromatase in human luteinized granulosa cells after controlled ovarian hyperstimulation

To examine expression of androgen receptor (AR), AR cofactors, estrogen (E) receptor alpha, E receptor beta, progesterone receptor, steroid receptor coactivator-1, and aromatase in human luteinized granulosa cells collected during oocyte retrieval. Prospective real-time reverse transcriptase-polymerase chain reaction study. Academic medical center. A total of 198 samples were brought into the study. Patients underwent the long protocol for assisted reproductive technology. Luteinized granulosa cells were collected transvaginally with ultrasound guidance. Quantitative reverse transcriptase-polymerase chain reaction was performed to quantify the mRNA expression of the investigated genes. The expression levels were determined as ratios between the studied genes and the reference gene beta-actin. There is little AR expression in human luteinized granulosa cells immediately preceding ovulation under controlled ovarian hyperstimulation. All aspirated follicles, despite their antral size, displayed a similar mRNA expression of the investigated genes in the luteinized granulosa cells. This study supports the possibility of a transition of androgen action from being an enhancer of follicular differentiation (through the AR) to being a substrate of E synthesis (through aromatase) at the time of oocyte retrieval. The present study also demonstrates no effect of follicular size upon the status of steroid receptor mRNA expression in the luteinized granulosa cells when follicles were at least >1.5 mL.

Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle

Prostaglandin E2 (PGE2) has been identified as the key ovulatory PG in the primate follicle. Follicular PGE2 levels increase just before the expected time of ovulation, suggesting that the midcycle LH surge induces the expression of enzymes involved in PGE2 synthesis. To identify the specific form(s) of prostaglandin E synthase (PGES) expressed by the primate periovulatory follicle, we examined granulosa and theca cell expression of the three microsomal (m) and cytosolic (c) forms of PGES (mPGES-1, mPGES-2 and cPGES) identified to date. Monkey granulosa cells and whole monkey ovaries were obtained from animals receiving exogenous gonadotropins to stimulate multiple follicular development; monkeys then received an ovulatory dose of HCG to initiate periovulatory events. Expression of mPGES-1 mRNA and protein by granulosa cells of periovulatory follicles increased in response to HCG administration, peaking just before the expected time of ovulation. Immunocytochemistry showed that mPGES-1 protein was present in both granulosa and theca cells of monkey periovulatory follicles. Monkey granulosa cells also expressed mPGES-2 and cPGES mRNA, but mRNA levels did not change in response to HCG administration. Isolated monkey theca cells expressed both mPGES-1 and cyclooxygenase-2 mRNA, and produced PGE2 in vitro. Human granulosa-lutein cells obtained from women undergoing treatment for infertility expressed mRNAs for mPGES-1, mPGES-2 and cPGES. These data indicate that mPGES-1 is a gonadotropin-regulated PG synthesis enzyme expressed by granulosa cells of primate periovulatory follicles and suggest that mPGES-1 may be the primary PGES responsible for the increased follicular PGE2 levels necessary for primate ovulation.

Spatiotemporal Expression of Heparanase During Human and Rodent Ovarian Folliculogenesis1

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs), major components of the basement membrane (BM) and extracellular matrix (ECM). Heparanase activity results in release of HSPG-bound molecules, including basic fibroblast growth factor (FGF2). Structural and functional development of the corpus luteum (CL) involves tissue remodeling, active angiogenesis, and steroid production. Heparanase-induced ECM and BM breakdown as well as FGF2-stimulated endothelial proliferation may have an important role in the regulation of luteal function. Heparanase mRNA was detected by reverse-transcription-polymerase chain reaction in granulosa cells recovered from follicular fluid of in vitro fertilization patients. Using sulfate-labeled ECM, heparanase enzymatic activity was determined in human luteinized granulosa cells. Employing immunohistochemistry, heparanase protein was localized predominantly in the theca interna cell layer of the mature antral follicle, whereas in human corpora lutea, both luteinized granulosa and theca cells were immunostained for heparanase. During luteolysis, heparanase was identified in macrophages surrounding the forming corpus albicans. In serially sectioned ovaries from unstimulated rats as well as from eCG-treated rats, expression of heparanase was noted exclusively in the ovarian steroid-producing interstitial tissue. Following an ovulatory dose of hCG, heparanase was immunostained also in lutein cells of the forming corpora lutea. Temporal expression of heparanase in granulosa cells during the luteal phase and in macrophages during luteal regression supports the hypothesis that heparanase plays a role in human ovarian ECM remodeling and may potentiate cellular migration and growth factor bioavailability.

Brown Kelp Modulates Endocrine Hormones in Female Sprague-Dawley Rats and in Human Luteinized Granulosa Cells

Epidemiological studies suggest that populations consuming typical Asian diets have a lower incidence of hormone-dependent cancers than populations consuming Western diets. These dietary differences have been mainly attributed to higher soy intakes among Asians. However, studies from our laboratory suggest that the anti-estrogenic effects of dietary kelp also may contribute to these reduced cancer rates. As a follow-up to previous findings of endocrine modulation related to kelp ingestion in a pilot study of premenopausal women, we investigated the endocrine modulating effects of kelp (Fucus vesiculosus) in female rats and human luteinized granulosa cells (hLGC). Kelp administration lengthened the rat estrous cycle from 4.3 +/- 0.96 to 5.4 +/- 1.7 d at 175 mg . kg(-1) body wt . d(-1) (P = 0.05) and to 5.9 +/- 1.9 d at 350 mg . kg(-1) . d(-1) (P = 0.002) and also led to a 100% increase in the length of diestrus (P = 0.02). Following 175 mg . kg(-1) . d(-1) treatment for 2 wk, serum 17beta-estradiol levels were reduced from 48.9 +/- 4.5 to 40.2 +/- 3.2 ng/L (P = 0.13). After 4 wk, 17beta-estradiol levels were reduced to 36.7 +/- 2.2 ng/L (P = 0.02). In hLGC, 25, 50, and 75 micromol/L treatment reduced 17beta-estradiol levels from 4732 +/- 591 to 3632 +/- 758, 3313 +/- 373, and 3060 +/- 538 ng/L, respectively. Kelp treatment also led to modest elevations in hLGC culture progesterone levels. Kelp extract inhibited the binding of estradiol to estrogen receptor alpha and beta and that of progesterone to the progesterone receptor, with IC(50) values of 42.4, 31.8, and 40.7 micromol/L, respectively. These data show endocrine modulating effects of kelp at relevant doses and suggest that dietary kelp may contribute to the lower incidence of hormone-dependent cancers among the Japanese.